The factors underlying the survival and maintenance of interstitial cells of Cajal (ICC) are not well understood. Loss of ICC is often associated with loss of neuronal nitric oxide synthase (nNOS) in humans, suggesting a possible link. The aim of this study was to determine the effect of neuronal NO on ICC in the mouse gastric body. The volumes of ICC were determined in nNOS−/− and control mice in the gastric body and in organotypic cultures using immunohistochemistry, laser scanning confocal microscopy and three‐dimensional reconstruction. ICC numbers were determined in primary cell cultures after treatment with an NO donor or an NOS inhibitor. The volumes of myenteric c‐Kit‐immunoreactive networks of ICC from nNOS−/− mice were significantly reduced compared with control mice. No significant differences in the volumes of c‐Kit‐positive ICC were observed in the longitudinal muscle layers. ICC volumes were either decreased or unaltered in the circular muscle layer after normalization for the volume of circular smooth muscle. The number of ICC was increased after incubation with S‐nitroso‐N‐acetylpenicillamine and decreased by N(G)‐nitro‐l‐arginine. Neuronally derived NO modulates ICC numbers and network volume in the mouse gastric body. NO appears to be a survival factor for ICC.